Inhibition of SERCA Ca²⁺ pumps by 2‐aminoethoxydiphenyl borate (2‐APB)

Abstract

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca 2+ channels and store-operated Ca 2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) Ca 2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca 2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca 2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC 50 values for inhibition being 325 and 725 lM, respectively, measured at pH 7.2). The Ca 2+ -ATPase is also more potently inhibited at lower pH (IC 50 ¼ 70 lM for SERCA 1A at pH 6). 2-APB decreases the affinity for Ca 2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca 2+ -ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca 2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca 2+ to their binding sites.Keywords: 2-APB; Ca 2+ -ATPase; Inhibition; SERCA.Ca 2+ plays a very important role in a number of signalling pathways, both within and between cells. The modulation of its levels in the cytosol is crucial to the viability and survival of the cell. Prolonged exposure to Ca 2+ can result in apoptosis, whereas a lack of rise in cytosolic [Ca 2+ ] may lead to the failure of signal transduction [1]. Specific pharmacological agents have been of great use as probes to aid our understanding of Ca 2+ signalling processes [2][3][4]. One such agent, 2-aminoethoxydiphenylborate (2-APB), has been reported to be a membrane permeable inhibitor of the inositol-1,4,5-trisphosphate (InsP 3 )-sensitive Ca 2+ channel with an IC 50 value of 42 lM (in the presence of 100 nM InsP 3 ) [5]. However, the effectiveness of 2-APB as a modulator of the InsP 3 receptor (InsP 3 R) has recently been questioned. We have recently shown that 2-APB is a lower affinity inhibitor of the type 1 InsP 3 R than was originally reported [6]. Our results show that the potency of 2-APB to inhibit InsP 3 -induced Ca 2+ release is dependent upon InsP 3 concentration used. At 0.25 lM InsP 3 , an IC 50 value of 220 lM was observed, while at 10 lM InsP 3 , the concentration of 2-APB required to half maximally inhibit Ca 2+ release is 1 mM.2-APB and xestospongin C (another cell permeant InsP 3 receptor inhibitor) have been used to characterize the mechanism of store-operated Ca 2+ entry, whereby Ca 2+ influx from the extracellular matrix is triggered by the emptying of Ca 2+ stores [7][8][9][10]. The concentrations of 2-APB used in these studies were in the ...

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels

“…3E). It is therefore surprising that 100 M 2-APB was able to completely inhibit re- We did not investigate the effects of 2-APB concentrations higher than 100 M since they caused a pronounced increase of basal Ca 2+ cyt , consistent with earlier reports of SERCA inhibition [11,26]. Concentrations of 2-APB lower than 100 M produced curious effects.…”

“…Furthermore, 2-APB rapidly blocks SOC by acting at extracellular sites [19,20,25]. In addition, it has been shown to inhibit sarco-endoplasmic reticulum Ca 2+ ATPases (SERCAs) [11,26], thus promoting the gradual release of Ca 2+ stores.…”

Membrane-delimited Regulation of Novel Background K+ Channels by MgATP in Murine Immature B Cells

“…After the initial suggestion as a membrane-permeable blocker of inositol 1,4,5-trisphosphate receptors (31), the blocking effects of 2-APB on the various membrane channel proteins, including Ca 2ϩ release-activated calcium channel, gap junction, and sarco/endoplasmic reticulum Ca 2ϩ -ATPase, have been reported (17,(31)(32)(33). On the other hand, depending on the concentration used, 2-APB facilitates the cytosolic regulator of adenylyl cyclase or other Ca 2ϩ -permeable cation channels (18,34,35).…”

TRPC1-mediated Inhibition of 1-Methyl-4-phenylpyridinium Ion Neurotoxicity in Human SH-SY5Y Neuroblastoma Cells

“…Because these studies were performed on isolated mitochondria, 2APB could function differently. Further, 2APB not only affects store-operated Ca 2ϩ entry channels (39) but also affects other proteins, such as the sarco-endoplasmic reticulum Ca 2ϩ ATPase pump (40), the voltage-dependent K ϩ channels (41), gap junctions (42), and TRPV channels (43). Interestingly, in Jurkat T cells 2-APB inhibits Ca 2ϩ efflux from mitochondria, thereby accumulating Ca 2ϩ that could activate proapoptotic proteins leading to cell death (44).…”